Methanol-utilizing yeasts use methanol as a carbon and energy source. The methanol utilization pathway begins with the oxidation of methanol to formaldehyde. That oxidation step is catalyzed by the enzyme alcohol oxidase (AOX, EC 1.1.3.13). While AOX is expressed at low levels in glucose-containing media, AOX accounts for 30% of solubilized intracellular proteins in methanol-containing media.
Pichia pastoris, a methanol-utilizing yeast, carries two AOX genes (the AOX1 gene and the AOX2 gene).
The AOX1 promoter and AOX2 promoter differ markedly in activity. AOX activity in Pichia pastoris is due mostly to expression at the AOX1 locus [Molecular and Cellular Biology, 9, 1316 (1989)]. Strains of Pichia pastoris in which a heterologous gene is substituted at the AOX1 locus grow slowly in methanol-containing media, theyby requiring a lengthy cultivation period, because the methanol utilization is based on AOX2 activity alone.
Among the two AOX genes coding for AOX, the AOX1 promoter, which is the regulatory region of the AOX1 gene, has high activity and can be used for the expression of heterologous proteins in high yields in methanol-utilizing yeasts. However, the AOX2 promoter is weak in activity and hence unsuitable for the expression of heterologous proteins.
Recently, studies have been conducted to produce heterologous proteins using the regulatory region of said AOX gene [e.g. EP-A-344459 (JP-A-2-104290) (the term "JP-A" used herein means an unexamined published Japanese patent application.); EP-A-343388 (JP-A-2-242694); GB-A-2217332 (JP-A2-303497)].
Natural AOX2 promoter is described in U.S. Pat. No. 5,032,516.
Once a gene coding for a heterologous protein has been inserted by substitution into the AOX1 gene region of the host, methanol utilization must depend solely on AOX2 activity. The growth rate of such mutants in methanol-containing media is inferior to that of the wild-type host which in return disadvantageously makes it necessary to employ a prolonged cultivation period.
It is an object of the invention to provide a yeast strain capable of good growth in methanol-containing media in contrast to other yeast strains lacking AOX1 gene activity and capable of expressing a heterologous protein, as well as a method of obtaining such a strain.
Another object of the invention is to provide a novel promoter for high-level expression of a heterologous protein, as well as a method of obtaining such promoter.
A further object is to provide a transformant comprising a plasmid containing said promoter and thereby establishing a heterologous protein production system.